The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.